This invention relates to the synthesis of oligonucleotides containing a (6-4) photoproduct, a lesion at base moieties generated by irradiation of DNA in vivo with UV light. Such lesions in DNA induce genetic mutations and cause cellular death and transformation. In spite of such a risk, normal transmission of genetic information is generally maintained because organisms have some DNA-repairing systems in cells (Annu. Rev. Biochem., Vol. 65, pp. 135 to 167 (1996)). Not only the (6-4) photoproduct synthesized by the process of the present invention and DNA containing the same can be used in studies for elucidating the mechanisms of mutations and repair of DNA, but also they are useful as a reagent for clinical tests, such as production of antibodies detecting damaged DNA.
When DNA is irradiated with UV light, two types of major lesions are formed at the sites of adjacent pyrimidine bases. One of them is the cis-syn cyclobutane pyrimidine dimer, and the other is the pyrimidine (6-4) pyrimidone photoproduct (hereinafter referred to as "a (6-4) photo-product"). It has been known that the (6-4) photo-product in DNA has the following structure and causes mutations with high frequency (see Proc. Natl. Acad. Sci. USA, vol. 88, pp. 9685 to 9689 (1991) and J. Mol. Biol., vol. 235, pp. 465 to 471 (1994)). ##STR3##
In previous studies, plasmid DNA irradiated with UV light has been used for the experiments of mutation and repair of DNA. However, it has been reported that various lesions including formamidopyrimidines are generated by UV light (Biochemistry, vol. 34, pp. 737 to 742 (1995)). Therefore, for detailed studies, it is necessary to use DNA having a specific lesion at a specific single site.
In previous reports on the preparation of damaged DNA, extremely short DNA fragments containing adjacent pyrimidines only at one site were irradiated with UV light, and from the reaction mixture, desired DNA containing the (6-4) photoproduct was purified by HPLC (high performance liquid chromatography) (J. Biol. Chem., vol. 268, pp. 11143 to 11151 (1993)). However, this process has the following disadvantages which reduce its practicality. Firstly, DNA obtained by this process has large limitations in the chain length and the base sequence, and its length is limited up to about a decamer. Secondly, the yield of the desired DNA is extremely low. Thirdly, the reaction mixture contains a Dewar isomer which is isomerized from the (6-4) photoproduct by exposure to the near UV light, and separation of this isomer is difficult (J. Biol. Chem., vol. 268, pp. 11143 to 11151 (1993)).